We aim to identify specific polypeptides involved (a) in transport of glucose-6-phosphate from the cytoplasmic to the lumenal side of endoplasmic reticulum of liver cells; (b) in hydrolysis of the substrate indicated. Our further intention would be to incorporate appropriate peptides into liposomes and reconstitute overall glucose-6-phosphatase activity (translocation and hydrolysis). Such a model system could be very informative. Point (a) above will be attacked by using tritiated diisothiocyanodiphenylethane disulfonic acid (3H2-DIDS) a potent inhibitor of transport of glucose-6-phosphate in intact microsomes, which also binds to a polypeptide of m.w. 54,000 specifically and covalently. The polypeptide so labeled is readily separated by gel electrophoresis and quantified by densitometry after autoradiography of the gel. Point (b) will be attacked by isolating the hydrolase polypeptide that becomes phosphorylated on a histidine by transfer of phosphate from glucose-6-phosphate, i.e., as an enzyme intermediate. Techniques very similar to those mentioned will be used. We shall attack the question of whether the translocase exerts control over the overall glucose-6-phosphatase reaction. The amount of 54,000 dalton polypeptide in liver cells from rats in different physiological states (e.g. diabetic, steroid treated) would be an indication of such control, an can be determined by 3H-DIDS labeling coupled with gel electrophoresis. In the case of brain microsomes (vesicles of endoplasmic reticulum) the situation is different from that of liver: evidence suggests that there may be no translocase. Latency to substrate is very high, and we shall attempt to explain how substrate reaches the hydrolase in this case. Glucose-6-phosphatase activity is the last step in gluconeogenesis and glycogenolysis. Our studies related to control of release of free glucose in health and in such diseases as diabetes. They also relate to brain function since we have previously found that cerebral glucose-6-phosphatase is involved in slow-wave sleep7